Coordinated control of XerC and XerD catalytic activities during Holliday junction resolution.

Site-specific recombinases XerC and XerD function in the segregation of circular bacterial replicons. In a recombining nucleoprotein complex containing two molecules each of XerC and XerD, coordinated reciprocal switches in recombinase activity ensure that only XerC or XerD is active at any one time. Mutated recombinases that carry sub?stitutions of a catalytic arginine residue stimulate cleavage and strand exchange mediated by the partner recombinase on DNA substrates that are normally recombined poorly by the partner. This is associated with a reciprocal impairment of the recombinase's own ability to initiate catalysis. The extent of this switch in catalysis is modulated by changes in recombination site sequence and is not a direct consequence of any catalytic defect. We propose that altered interactions between the mutated proteins and their wild-type partners lead to an increased level of an alternative Holliday junction intermediate that has a conformation appropriate for resolution by the partner recombinase. The results indicate how subtle changes in protein-DNA architecture at a Holliday junction can redirect recombination outcome.

Reciprocal control of catalysis by the tyrosine recombinases XerC and XerD: an enzymatic switch in site-specific recombination.

In Xer site-specific recombination, sequential DNA strand exchange reactions are catalyzed by a heterotetrameric complex composed of two recombinases, XerC and XerD. It is demonstrated that XerC and XerD catalytic activity is controlled by an interaction involving the C-terminal end of each protein (the donor region) and an internal region close to the active site (the acceptor region). Mutations in these regions reciprocally alter the relative activity of XerC and XerD, with their combination producing synergistic effects on catalysis. The data support a model in which C-terminal intersubunit interactions contribute to coupled protein-DNA conformational changes that lead to sequential activation and reciprocal inhibition of pairs of active sites in the recombinase tetramer during recombination.

Crystal structure of the site-specific recombinase, XerD.

The structure of the site-specific recombinase, XerD, that functions in circular chromosome separation, has been solved at 2.5 A resolution and reveals that the protein comprises two domains. The C-terminal domain contains two conserved sequence motifs that are located in similar positions in the structures of XerD, lambda and HP1 integrases. However, the extreme C-terminal regions of the three proteins, containing the active site tyrosine, are very different. In XerD, the arrangement of active site residues supports a cis cleavage mechanism. Biochemical evidence for DNA bending is encompassed in a model that accommodates extensive biochemical and genetic data, and in which the DNA is wrapped around an alpha-helix in a manner similar to that observed for CAP complexed with DNA.

Action of site-specific recombinases XerC and XerD on tethered Holliday junctions.

In Xer site-specific recombination, two related recombinases, XerC and XerD, mediate the formation of recombinant products using Holliday junction-containing DNA molecules as reaction intermediates. Each recombinase catalyses the exchange of one pair of specific strands. By using synthetic Holliday junction-containing recombination substrates in which two of the four arms are tethered in an antiparallel configuration by a nine thymine oligonucleotide, we show that XerD catalyses efficient strand exchange only when its substrate strands are 'crossed'. XerC also catalyses very efficient strand exchange when its substrate strands are 'crossed', though it also appears to be able to mediate strand exchange when its substrate strands are 'continuous'. By using chemical probes of Holliday junction structure in the presence and absence of bound recombinases, we show that recombinase binding induces unstacking of the bases in the centre of the recombination site, indicating that the junction branch point is positioned there and is distorted as a consequence of recombinase binding.

Xer site-specific recombination in vitro.

Two related recombinases, XerC and XerD, belonging to the lambda integrase family of enzymes, are required for Xer site-specific recombination in vivo. In order to understand the roles of these proteins in the overall reaction mechanism, an in vitro recombination system using a synthetic Holliday junction-containing substrate has been developed. Recombination of this substrate is efficient and requires both XerC and XerD. However, only exchange of one pair of strands, the one corresponding to the conversion of the Holliday junction intermediate back to the substrate, has been observed. Recombination reactions using XerC and XerD derivatives that are mutant in their presumptive catalytic residues, or are maltose-binding fusion recombinase derivatives, have demonstrated that this pair of strand exchanges is catalysed by XerC. The site of XerC-mediated cleavage has been located to between the last nucleotide of the XerC binding site and the first nucleotide of the central region. Cleavage at this site generates a free 5'-OH and a covalent complex between XerC and the 3' end of the DNA.

Site-specific recombination and circular chromosome segregation.

The Xer site-specific recombination system functions in Escherichia coli to ensure that circular plasmids and chromosomes are in the monomeric state prior to segregation at cell division. Two recombinases, XerC and XerD, bind cooperatively to a recombination site present in the E. coli chromosome and to sites present in natural multicopy plasmids. In addition, recombination at the natural plasmid site cer, present in ColEl, requires the function of two additional accessory proteins, ArgR and PepA. These accessory proteins, along with accessory DNA sequences present in the recombination sites of plasmids are used to ensure that recombination is exclusively intramolecular, converting circular multimers to monomers. Wild-type and mutant recombination proteins have been used to analyse the formation of recombinational synapses and the catalysis of strand exchange in vitro. These experiments demonstrate how the same two recombination proteins can act with different outcomes, depending on the organization of DNA sites at which they act. Moreover, insight into the separate roles of the two recombinases is emerging.

Two related recombinases are required for site-specific recombination at dif and cer in E. coli K12.

The stable inheritance of ColE1-related plasmids and the normal partition of the E. coli chromosome require the function of the Xer site-specific recombination system. We show that in addition to the XerC recombinase, whose function has already been implicated in this system, a second chromosomally encoded recombinase, XerD, is required. The XerC and XerD proteins show 37% identity and bind to separate halves of the recombination site. Both proteins act catalytically in the recombination reaction. Recombination site asymmetry and the requirement of two recombinases ensure that only correctly aligned sites are recombined. We predict that normal partition of most circular chromosomes requires the participation of site-specific recombination to convert any multimers (arising by homologous recombination) to monomers.

Purification of TnsB, a transposition protein that binds to the ends of Tn7.

We have purified TnsB, a transposition protein encoded by the bacterial transposon Tn7. The purification procedure involves three chromatographic steps (DNA-cellulose, norleucine-Sepharose, and phosphocellulose) and yields milligram quantities of highly purified protein. The apparent molecular mass of denatured TnsB protein is approximately 85 kDa. Gel filtration chromatography and sucrose gradient sedimentation studies indicate that in solution, native TnsB is a monomer of nonspherical shape. Using DNase I protection analysis, we established that TnsB is a sequence-specific DNA-binding protein that recognizes multiple sites in both ends of the transposon. The TnsB binding sites, three in the left end of Tn7 and four in the right end, are highly related in nucleotide sequence and are located in DNA segments that we have previously shown contain cis-acting sequences important for Tn7 transposition. Our results also show that one of the TnsB binding sites overlaps a proposed promoter for the transposition genes of Tn7. These studies suggest that the specific binding of TnsB to the ends of Tn7 mediates recombination and may also regulate the expression of Tn7-encoded transposition genes.

Interaction of the Tn7-encoded transposition protein TnsB with the ends of the transposon.

We have used several high resolution methods to examine the interaction of TnsB, a transposition protein encoded by the bacterial transposon Tn7, with its binding sites at the ends of the transposon. These binding sites lie within the DNA segments that are directly involved in transposition. We show that the binding of TnsB to DNA can promote DNA bending, suggesting that the interaction of TnsB with the ends may result in formation of a highly organized protein-DNA complex. We also identify likely positions of close contact between of TnsB and its binding sites. Analysis of the interaction of TnsB with intact Tn7 ends reveals TnsB occupies its binding sites in a particular order, the sites immediately adjacent to the transposon termini being occupied only after other inner sites are bound. Such ordered occupancy suggests that the various binding sites have differing apparent affinities for TnsB.

Transposon Tn7. cis-Acting sequences in transposition and transposition immunity.

We have identified and characterized the cis-acting sequences at the termini of the bacterial transposon Tn7 that are necessary for its transposition. Tn7 participates in two kinds of transposition event: high-frequency transposition to a specific target site (attTn7) and low-frequency transposition to apparently random target sites. Our analyses suggest that the same sequences at the Tn7 ends are required for both transposition events. These sequences differ in length and nucleotide structure: about 150 base-pairs at the left end (Tn7L) and about 70 base-pairs at the right end (Tn7R) are necessary for efficient transposition. We also show that the ends of Tn7 are functionally distinct: a miniTn7 element containing two Tn7R ends is active in transposition but an element containing two Tn7L ends is not. We also report that the presence of Tn7's cis-acting transposition sequences anywhere in a target replicon inhibits subsequent insertion of another copy of Tn7 into either an attTn7 target site or into random target sites. The inhibition to an attTn7 target site is most pronounced when the Tn7 ends are immediately adjacent to attTn7. We also show that the presence of Tn7R's cis-acting transposition sequences in a target replicon is necessary and sufficient to inhibit subsequent Tn7 insertion into the target replicon.

Identification of a transposon Tn7-dependent DNA-binding activity that recognizes the ends of Tn7.

The bacterial transposon Tn7 is distinguished by its capacity for high-frequency transposition to a specific site in the Escherichia coli chromosome. tnsB is one of the five Tn7-encoded transposition genes. We have identified in vitro a tnsB-dependent DNA binding activity that interacts specifically with cis-acting transposition sequences at the Tn7 termini. Although the left and right termini of Tn7 are structurally distinct, each end contains several copies of a closely homologous 22-base-pair sequence. We present results indicating that this 22-base-pair repeat sequence is recognized by the tnsB-dependent binding activity.

The antimutagenic effect of selenium on 7,12-dimethylbenz(a)anthracene and metabolites in the amesSalmonella/microsome system.

The antimutagenic effect of selenium as sodium selenite, sodium selenate, selenium dioxide, and seleno-methionine was studied in the AmesSalmonella/microsome mutagenicity test using 7,12-dimethylbenz(a)anthracene (DMBA) and some of its metabolites. Selenium (20 ppm) as sodium selenite reduced the number of histidine revertants on plates containing up to 100 µg DMBA/plate. Increasing concentrations of selenium as sodium selenite, sodium selenate, and selenium dioxide up to 40 ppm Se progressively decreased the number of revertants caused by 50 µg DMBA. DMBA and its metabolites 7-hydroxymethyl-12-methylbenz(a)anthracene, 12-hydroxymethyl-7-methylbenz(a)anthracene, and 3-hydroxy-7,12-dimethylbenz(a)anthracene were mutagenic forSalmonella typhimurium TA100 in the presence of an S-9 mixture. Selenium supplementation as Na2SeO3 reduced the number of revertants induced by these metabolites to background levels. The antimutagenic effect of inorganic selenium compounds cannot be explained by toxicity of selenium as determined by viability tests withSalmonella typhimurium TA100. Selenium supplementation in all forms examined, except sodium selenate, decreased the rate of spontaneous reversion. Selenium as sodium selenate was slightly mutagenic at concentrations of 4 ppm or less. Higher concentration of Na2SeO4 inhibited the mutagenicity of DMBA. The present studies support the anticarcinogenic potential of selenium and indicate that form and concentration are important factors in this trace element's efficacy.